Although the uterine vascular effects of estrogen have been studied for over 70 years, the mechanism by which estrogen produces vasodilation remains unclear. Our laboratory was the first to show that a significant component of the uterine response to estrogen in the nonpregnant sheep is mediated by the release of nitric oxide (NO). We and others have shown that estradiol-17beta increases the expression and activity of endothelial nitric oxide synthase (eNOS) in the uterine circulation. However, it is not currently clear how this occurs or if, in addition to eNOS, neuronal nitric oxide synthase (nNOS) or inducible nitric oxide synthase (iNOS) are also important in maintaining the sustained vasodilatory response seen after estrogen administration. Furthermore, it is not clear how estrogen modulates these NOS isoforms at the cellular and molecular level. Recently a new estrogen receptor, ERbeta has been isolated and emerging data suggest that this receptor may mediate a significant portion of the effects of estrogen in the vasculature. We hypothesize that uterine vasodilation produced by estradiol-17beta is mediated by specific interaction with both ERalpha and ERbeta, which subsequently activates eNOS (and potentially nNOS) via a nongenomic pathway, and iNOS via a genomic pathway, leading to increases in NO. The present application plans to evaluate the role of ER as a modulator of the NOS isoforms in the uterine circulation using a combination of physiologic and molecular endpoints. We will monitor the uterine hemodynamic responses to locally and systemically administered pharmacological antagonists that are selective for specific isoforms of NOS using a well-characterized ovine model. We intend to evaluate the expression of ERalpha and ERbeta in the ovine uterine vasculature and explore how estrogen alters eNOS, nNOS and NOS expression in endothelial cells and vascular smooth muscle. Finally we plan to determine if endogenous estrogen, acting through the uterine vascular NOS system, plays a critical role in increasing and maintaining uterine blood flow in late pregnancy. We believe that the information obtained in this revised application will provide new and important understanding into the mechanisms regulating vascular tone and hemodynamics in the uterine circulation in both the nonpregnant and pregnant animal.